JP2015511819A - 分子計数のための組成物およびキット - Google Patents
分子計数のための組成物およびキット Download PDFInfo
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Abstract
Description
本願は、2012年2月27日に出願した米国仮出願第61/603,921号および2012年12月21日に出願した米国仮出願61/745,385号の利益を主張する。これらの両方が、その全体に、本明細書に参考として援用される。
一部の実施形態では、本明細書に、RNA分子のデジタル式逆転写のための方法、キット、およびシステムを開示する。一部の場合には、方法は、(a)(i)複数のRNA分子が、少なくとも2つの異なる配列を含むRNA分子を2つ以上含み、(ii)複数のオリゴヌクレオチドタグが、2つ以上の異なる一意識別子配列を含むオリゴヌクレオチドタグを含む、複数のRNA分子を含む試料を複数のオリゴヌクレオチドタグと接触させることにより標識RNA分子を生成するステップと、(b)標識RNA分子を逆転写酵素と接触させて第1の鎖合成反応を実行することによって標識cDNA分子を生成するステップと、(c)標識cDNA分子を固体支持体にハイブリダイズさせることによって標識cDNA分子を検出するステップとを含む。
水 7.8μl
K562 全RNA(1μg/μl) 1μl
10mM dNTP 1μl
遺伝子特異的dUTPプライマー(10μM) 0.4μl
確率的標識(10μM) * 0.4μl
合計 10.6μl
5×第1の鎖緩衝剤 4μl
0.1M DTT 1μl
Super RNアーゼIn(20U/μl) 1μl
MMLV RT 1μl
NEB Taqポリメラーゼ 0.4μl
スーパースクリプトIIIおよびチタニウムTaqの代わりにMMLV RTおよびNEB Taqポリメラーゼの使用を、代替的に使用することができる。
37°60分間、その後、
94°2分間
55°2分間
68°2分間
を3サイクル、
次いで、永久に4°
ヌクレアーゼフリー水 10.9μl
10×NEB Taq緩衝剤 1.5μl
10mM dNTP 0.3μl
遺伝子特異的プライマー(1μM) 1μl
汎用PCRプライマー(1μM)* 1μl
NEB Taqポリメラーゼ 0.3μl
合計 15μl
94°2分間、その後、
94°2分間
55°2分間
68°2分間
を30サイクル、
次いで、68°4分間
永久に4°
ヌクレアーゼフリー水 39.5μl
10×NEB Taq緩衝剤 5μl
10mM dNTP 1μl
遺伝子特異的ネステッドプライマー(10μM) 1μl
5Tye 563標識汎用PCRプライマー(10μM)* 1μl
NEB Taqポリメラーゼ 0.5μl
合計 48μl
94°2分間、その後、
94°2分間
55°2分間
68°2分間
を30サイクル、
次いで、68°4分間
永久に4°
洗浄液A(6×SSPE+0.01%Triton X−100) 55μl
Cy3対照オリゴ(760pM)* 1μl
PCR生成物 20μl
合計 76μl
Claims (163)
- a.複数のRNA分子を含む試料を複数のオリゴヌクレオチドタグと接触させて標識RNA分子を生成するステップであって、
i.前記複数のRNA分子は異なる配列の少なくとも2つのmRNA分子を含み、
ii.前記複数のオリゴヌクレオチドタグは異なる配列の少なくとも2つのオリゴヌクレオチドタグを含み、
iii.前記複数のオリゴヌクレオチドタグはオリゴdT配列を含む、ステップと、
b.前記標識RNA分子を逆転写酵素と接触させて第1の鎖合成反応を実行することによって標識cDNA分子を生成するステップと、
c.前記標識cDNA分子を固体支持体とハイブリダイズすることによって前記標識cDNA分子を検出するステップと
を含むデジタル式逆転写方法。 - 前記オリゴヌクレオチドタグが一意識別子領域をさらに含む、請求項1に記載の方法。
- 前記一意識別子領域が少なくとも1ヌクレオチドの長さである、請求項2に記載の方法。
- 前記オリゴヌクレオチドタグが汎用プライマー結合部位をさらに含む、請求項1に記載の方法。
- 前記オリゴヌクレオチドタグが少なくとも1ヌクレオチドの長さである、請求項1に記載の方法。
- 前記固体支持体がアレイである、請求項1に記載の方法。
- 前記固体支持体がアドレス可能なアレイである、請求項1に記載の方法。
- 前記固体支持体がAffymetrix 3Kタグアレイ、Arrayjet非接触印刷アレイまたはApplied Microarrays Inc(AMI)アレイである、請求項1に記載の方法。
- 前記固体支持体がビーズである、請求項1に記載の方法。
- ステップ(b)の前記標識cDNA分子に対してポリメラーゼ連鎖反応を実行して二本鎖標識cDNA分子を生成することをさらに含む、請求項1に記載の方法。
- 前記ポリメラーゼ連鎖反応を実行することが、第1の標的特異的プライマーを前記標識cDNA分子にアニールすることを含む、請求項10に記載の方法。
- 前記ポリメラーゼ連鎖反応を実行することが、汎用プライマーを前記オリゴヌクレオチドタグの前記汎用プライマー結合部位にアニールすることをさらに含む、請求項10に記載の方法。
- 前記ポリメラーゼ連鎖反応が絶対PCR、HD−PCR、次世代PCR、デジタル式RTAまたはその任意の組合せを含む、請求項10に記載の方法。
- 前記二本鎖標識cDNA分子に対してネステッドPCR反応を実行することをさらに含む、請求項10に記載の方法。
- 前記ネステッドPCR反応を実行することが、前記二本鎖標識cDNA分子を変性させて変性一本鎖標識cDNA分子を生成することを含む、請求項14に記載の方法。
- 前記ネステッドPCR反応を実行することが、第2の標的特異的プライマーを前記変性一本鎖標識cDNA分子にアニールすることをさらに含む、請求項14に記載の方法。
- 前記ネステッドPCR反応を実行することが、汎用プライマーを前記オリゴヌクレオチドタグの前記汎用プライマー結合部位にアニールすることをさらに含む、請求項14に記載の方法。
- 前記オリゴヌクレオチドタグの少なくとも一部、前記標識cDNA分子の少なくとも一部、その相補配列、その逆相補配列またはその任意の組合せの配列を決定するために、配列決定反応を実行することをさらに含む、請求項1に記載の方法。
- 前記標識cDNA分子を検出することが、アレイ検出器、蛍光リーダー、非蛍光検出器、CRリーダーまたはスキャナを含む、請求項1に記載の方法。
- 前記アレイ検出器がフラットベッドスキャナである、請求項19に記載の方法。
- 前記蛍光リーダーがSensovation,AGの蛍光リーダーである、請求項19に記載の方法。
- 前記標識cDNA分子を検出することが、前記固体支持体にハイブリダイズされた前記標識cDNA分子を検出することを含む、請求項1に記載の方法。
- ハイブリダイゼーション連鎖反応を実行することをさらに含む、請求項1に記載の方法。
- 前記オリゴヌクレオチドタグが1つまたは複数の二次構造を含む、請求項1に記載の方法。
- 前記1つまたは複数の二次構造がヘアピンである、請求項24に記載の方法。
- 前記オリゴヌクレオチドタグが線状である、請求項1に記載の方法。
- a.複数のオリゴヌクレオチドタグであって、前記複数のオリゴヌクレオチドタグのオリゴヌクレオチドタグは、
i.標的特異的領域、および
ii.一意識別子領域
を含むオリゴヌクレオチドタグと、
b.酵素と
を含むキット。 - 前記酵素が逆転写酵素である、請求項27に記載のキット。
- 前記酵素がリガーゼである、請求項27に記載のキット。
- 前記酵素がポリメラーゼである、請求項27に記載のキット。
- 前記酵素がRNアーゼである、請求項27に記載のキット。
- 前記酵素がDNアーゼである、請求項27に記載のキット。
- 前記酵素がエンドヌクレアーゼである、請求項27に記載のキット。
- 前記オリゴヌクレオチドタグが少なくとも25ヌクレオチドの長さである、請求項27に記載のキット。
- 前記一意識別子領域が少なくとも10ヌクレオチドの長さである、請求項27に記載のキット。
- 前記標的特異的領域が少なくとも10ヌクレオチドの長さである、請求項27に記載のキット。
- 前記標的特異的領域がオリゴdT配列を含む、請求項27に記載のキット。
- 前記オリゴヌクレオチドタグが汎用プライマー結合部位をさらに含む、請求項27に記載のキット。
- 支持体をさらに含む、請求項27に記載のキット。
- 前記支持体が半固体支持体である、請求項39に記載のキット。
- 前記支持体が固体支持体である、請求項39に記載のキット。
- 前記固体支持体がアレイである、請求項41に記載のキット。
- 前記支持体がアドレス可能なアレイである、請求項39に記載のキット。
- 前記支持体がAffymetrix 3Kタグアレイ、Arrayjet非接触印刷アレイまたはApplied Microarrays Inc(AMI)アレイである、請求項39に記載のキット。
- 前記支持体がビーズである、請求項39に記載のキット。
- プライマーをさらに含む、請求項27に記載のキット。
- 前記プライマーが汎用プライマーである、請求項46に記載のキット。
- 前記プライマーが前記オリゴヌクレオチドタグに結合する、請求項46に記載のキット。
- 前記プライマーが前記オリゴヌクレオチドタグの汎用プライマー結合部位に結合する、請求項46に記載のキット。
- 対照オリゴをさらに含む、請求項27に記載のキット。
- 前記対照オリゴが少なくとも15ヌクレオチドを含む、請求項50に記載のキット。
- 前記対照オリゴが明るいハイブリダイゼーション対照オリゴである、請求項50に記載のキット。
- 前記対照オリゴがスパイクイン鋳型対照である、請求項50に記載のキット。
- 前記オリゴヌクレオチドタグが標識をさらに含む、請求項27に記載のキット。
- 前記プライマーが標識をさらに含む、請求項46に記載のキット。
- 前記対照オリゴが標識をさらに含む、請求項50に記載のキット。
- 前記標識が色素標識である、請求項54〜56のいずれかに記載のキット。
- 前記標識がCy3色素である、請求項54〜56のいずれかに記載のキット。
- 前記標識がTye563色素である、請求項54〜56のいずれかに記載のキット。
- 緩衝剤をさらに含む、請求項27に記載のキット。
- 担体をさらに含む、請求項27に記載のキット。
- 洗浄剤をさらに含む、請求項27に記載のキット。
- サーマルサイクラーをさらに含む、請求項27に記載のキット。
- シーケンサーをさらに含む、請求項27に記載のキット。
- ハイブリダイゼーションチャンバーをさらに含む、請求項27に記載のキット。
- 検出器をさらに含む、請求項27に記載のキット。
- アレイ検出器、蛍光リーダー、非蛍光検出器、CRリーダーまたはスキャナをさらに含む、請求項27に記載のキット。
- 前記蛍光リーダーがSensovationまたはAGの蛍光リーダーである、請求項67に記載のキット。
- 前記スキャナがフラットベッドスキャナである、請求項67に記載のキット。
- コンピューターをさらに含む、請求項27に記載のキット。
- 前記コンピューターがメモリデバイスを含む、請求項70に記載のキット。
- 前記メモリデバイスがデータを保存することが可能である、請求項71に記載のキット。
- ソフトウェアプログラムをさらに含む、請求項27に記載のキット。
- コンピューター可読プログラムをさらに含む、請求項27に記載のキット。
- a.複数の分子を含む試料を複数のオリゴヌクレオチドタグと接触させて標識分子を生成するステップであって、
i.前記複数の分子は異なる配列の少なくとも2つの分子を含み、
ii.前記複数のオリゴヌクレオチドタグは異なる配列の少なくとも2つのオリゴヌクレオチドタグを含み、
iii.前記試料は少なくとも1つの細胞に由来する、ステップと、
b.前記標識分子を固体支持体とハイブリダイズすることによって前記標識分子を検出するステップと
を含む細胞分析方法。 - a.複数の分子を複数のオリゴヌクレオチドタグで確率的に標識して標識分子を生成するステップであって、
i.前記複数の分子は異なる配列の少なくとも2つの分子を含み、
ii.前記複数のオリゴヌクレオチドタグは異なる配列の少なくとも2つのオリゴヌクレオチドタグを含む、ステップと、
b.前記標識分子を増幅して標識アンプリコンを生成するステップと、
c.前記標識アンプリコンを検出するステップと
を含むクローン増幅方法。 - 前記オリゴヌクレオチドタグが一意識別子領域をさらに含む、請求項75〜76のいずれかに記載の方法。
- 前記一意識別子領域が少なくとも10ヌクレオチドの長さである、請求項77に記載の方法。
- 前記一意識別子領域が前記分子とハイブリダイズできない、請求項77に記載の方法。
- 前記オリゴヌクレオチドタグが汎用プライマー結合部位をさらに含む、請求項75〜76のいずれかに記載の方法。
- 前記オリゴヌクレオチドタグが少なくとも20ヌクレオチドの長さである、請求項75〜76のいずれかに記載の方法。
- 前記オリゴヌクレオチドタグが標的特異的領域をさらに含む、請求項75〜76のいずれかに記載の方法。
- 前記標的特異的領域がオリゴdT配列を含む、請求項82に記載の方法。
- 前記標的特異的領域が少なくとも10ヌクレオチドの長さである、請求項82に記載の方法。
- 前記固体支持体がアレイである、請求項75〜76のいずれかに記載の方法。
- 前記固体支持体がアドレス可能なアレイである、請求項75〜76のいずれかに記載の方法。
- 前記固体支持体がAffymetrix 3Kタグアレイ、Arrayjet非接触印刷アレイまたはApplied Microarrays Inc(AMI)アレイである、請求項75〜76のいずれかに記載の方法。
- 前記固体支持体がビーズである、請求項75〜76のいずれかに記載の方法。
- 第1の鎖合成反応を実行して標識cDNA分子を生成することをさらに含む、請求項75〜76のいずれかに記載の方法。
- 前記標識分子またはその任意の生成物に対してポリメラーゼ連鎖反応を実行して二本鎖標識分子を生成することをさらに含む、請求項75に記載の方法。
- 前記標識分子を増幅することがポリメラーゼ連鎖反応を実行することを含む、請求項76に記載の方法。
- 前記ポリメラーゼ連鎖反応を実行することが、第1の標的特異的プライマーを前記標識分子またはその任意の生成物にアニールすることを含む、請求項90〜91のいずれかに記載の方法。
- 前記ポリメラーゼ連鎖反応を実行することが、汎用プライマーを前記オリゴヌクレオチドタグの前記汎用プライマー結合部位にアニールすることをさらに含む、請求項90〜91のいずれかに記載の方法。
- 前記ポリメラーゼ連鎖反応が絶対PCR、HD−PCR、次世代PCR、デジタル式RTAまたはその任意の組合せを含む、請求項90〜91のいずれかに記載の方法。
- 前記二本鎖標識cDNA分子に対してネステッドPCR反応を実行することをさらに含む、請求項75〜76のいずれかに記載の方法。
- 前記ネステッドPCR反応を実行することが、前記標識分子またはその任意の生成物を変性させて変性一本鎖標識分子またはその任意の生成物を生成することを含む、請求項95に記載の方法。
- 前記ネステッドPCR反応を実行することが、第2の標的特異的プライマーを前記変性一本鎖標識分子またはその任意の生成物にアニールすることをさらに含む、請求項95〜96のいずれかに記載の方法。
- 前記ネステッドPCR反応を実行することが、汎用プライマーを前記オリゴヌクレオチドタグの前記汎用プライマー結合部位にアニールすることをさらに含む、請求項95〜96のいずれかに記載の方法。
- 前記オリゴヌクレオチドタグの少なくとも一部、前記標識分子の少なくとも一部、その生成物、その相補配列、その逆相補配列またはその任意の組合せの配列を決定するために、配列決定反応を実行することをさらに含む、請求項75〜76のいずれかに記載の方法。
- 前記標識分子またはその任意の生成物を検出することが、アレイ検出器、蛍光リーダー、非蛍光検出器、CRリーダーまたはスキャナを含む、請求項75〜76のいずれかに記載の方法。
- 前記蛍光リーダーがSensovation、またはAGの蛍光リーダーである、請求項100に記載の方法。
- 前記スキャナがフラットベッドスキャナである、請求項100に記載の方法。
- 前記標識分子またはその任意の生成物を検出することが、前記固体支持体にハイブリダイズされた前記標識分子を検出することを含む、請求項75〜76のいずれかに記載の方法。
- 前記分子が核酸分子である、請求項75〜76のいずれかに記載の方法。
- 前記核酸分子がDNA分子である、請求項104に記載の方法。
- 前記核酸分子がRNA分子である、請求項104に記載の方法。
- 前記分子がペプチドである、請求項75〜76のいずれかに記載の方法。
- 前記ペプチドがポリペプチドである、請求項107に記載の方法。
- 前記複数の分子が細胞に由来する、請求項76に記載の方法。
- 前記試料が単一の細胞に由来する、請求項75に記載の方法。
- 前記試料が約100個未満の細胞に由来する、請求項75に記載の方法。
- 前記試料が約50個未満の細胞に由来する、請求項75に記載の方法。
- 前記試料が約20個未満の細胞に由来する、請求項75に記載の方法。
- 前記試料が約10個未満の細胞に由来する、請求項75に記載の方法。
- 前記試料が約5個未満の細胞に由来する、請求項75に記載の方法。
- 前記細胞が哺乳動物細胞である、請求項75に記載の方法。
- 前記細胞がヒト細胞である、請求項75に記載の方法。
- 前記細胞が疾患または状態を患っている対象に由来する、請求項75に記載の方法。
- 前記疾患または状態ががんである、請求項118に記載の方法。
- 前記疾患または状態が病原体感染症である、請求項118に記載の方法。
- 前記疾患または状態が遺伝的障害である、請求項118に記載の方法。
- 前記細胞が健康な対象に由来する、請求項109〜110のいずれかに記載の方法。
- 前記細胞が病的細胞である、請求項109〜110のいずれかに記載の方法。
- 前記病的細胞ががん性細胞である、請求項123に記載の方法。
- 前記細胞が健康な細胞である、請求項109〜110のいずれかに記載の方法。
- 前記細胞が病的細胞でも感染細胞でもない、請求項125に記載の方法。
- 標識分子が確率的標識によって生成される、請求項1および75〜76のいずれかに記載の方法。
- 1つまたは複数の核酸分子を複数のヘアピンオリゴヌクレオチドタグで確率的に標識するステップを含み、
(a)前記ヘアピンオリゴヌクレオチドタグはオーバーハングを含み、
(b)前記1つまたは複数の核酸分子はハイブリダイゼーション連鎖反応のための開始剤として作用する、
確率的標識に基づくハイブリダイゼーション連鎖反応方法。 - 前記ヘアピンオリゴヌクレオチドタグの少なくとも一部が前記1つまたは複数の核酸分子の少なくとも一部とハイブリダイズする、請求項128に記載の方法。
- 前記ヘアピンオリゴヌクレオチドタグがオリゴdT配列を含む、請求項128に記載の方法。
- 前記1つまたは複数の核酸分子が1つまたは複数のアダプターを含む、請求項128に記載の方法。
- 前記ヘアピンオリゴヌクレオチドタグの少なくとも一部が前記1つまたは複数のアダプターの少なくとも一部とハイブリダイズする、請求項131に記載の方法。
- 前記複数のヘアピンオリゴヌクレオチドタグの少なくとも1つのヘアピンオリゴヌクレオチドタグが1つまたは複数の標識を含む、請求項128に記載の方法。
- 前記複数のヘアピンオリゴヌクレオチドタグの少なくとも1つのヘアピンオリゴヌクレオチドタグが2つ以上の標識を含む、請求項128に記載の方法。
- 前記複数のヘアピンオリゴヌクレオチドタグの各ヘアピンオリゴヌクレオチドタグが1つまたは複数の標識を含む、請求項128に記載の方法。
- 前記複数のヘアピンオリゴヌクレオチドタグの各ヘアピンオリゴヌクレオチドタグが2つ以上の標識を含む、請求項128に記載の方法。
- 前記ヘアピンオリゴヌクレオチドタグが標識を含まない、請求項128に記載の方法。
- 前記複数のヘアピンオリゴヌクレオチドタグが、5’オーバーハングを有する1つまたは複数のヘアピンオリゴヌクレオチドタグ、3’オーバーハングを有するヘアピンオリゴヌクレオチドタグ、またはその組合せを含む、請求項128に記載の方法。
- 前記ヘアピンオリゴヌクレオチドタグのステム部分が1ヌクレオチド以上の長さである、請求項128に記載の方法。
- 前記ヘアピンオリゴヌクレオチドタグのステム部分が2ヌクレオチド以上の長さである、請求項128に記載の方法。
- 前記ヘアピンオリゴヌクレオチドタグのステム部分が3ヌクレオチド以上の長さである、請求項128に記載の方法。
- 前記ヘアピンオリゴヌクレオチドタグのステム部分が4ヌクレオチド以上の長さである、請求項128に記載の方法。
- 前記ヘアピンオリゴヌクレオチドタグのステム部分が5ヌクレオチド以上の長さである、請求項128に記載の方法。
- 前記ヘアピンオリゴヌクレオチドタグのステム部分が6ヌクレオチド以上の長さである、請求項128に記載の方法。
- 前記ヘアピンオリゴヌクレオチドタグのステム部分が7ヌクレオチド以上の長さである、請求項128に記載の方法。
- 前記ヘアピンオリゴヌクレオチドタグのステム部分が8ヌクレオチド以上の長さである、請求項128に記載の方法。
- 前記ヘアピンオリゴヌクレオチドタグのステム部分が9ヌクレオチド以上の長さである、請求項128に記載の方法。
- 前記ヘアピンオリゴヌクレオチドタグのステム部分が10ヌクレオチド以上の長さである、請求項128に記載の方法。
- 前記ヘアピンオリゴヌクレオチドタグのループ部分が1ヌクレオチド以上の長さである、請求項128に記載の方法。
- 前記ヘアピンオリゴヌクレオチドタグのループ部分が2ヌクレオチド以上の長さである、請求項128に記載の方法。
- 前記ヘアピンオリゴヌクレオチドタグのループ部分が3ヌクレオチド以上の長さである、請求項128に記載の方法。
- 前記ヘアピンオリゴヌクレオチドタグのループ部分が4ヌクレオチド以上の長さである、請求項128に記載の方法。
- 前記ヘアピンオリゴヌクレオチドタグのループ部分が5ヌクレオチド以上の長さである、請求項128に記載の方法。
- 前記ヘアピンオリゴヌクレオチドタグのループ部分が6ヌクレオチド以上の長さである、請求項128に記載の方法。
- 前記ヘアピンオリゴヌクレオチドタグのループ部分が7ヌクレオチド以上の長さである、請求項128に記載の方法。
- 前記ヘアピンオリゴヌクレオチドタグのループ部分が8ヌクレオチド以上の長さである、請求項128に記載の方法。
- 前記ヘアピンオリゴヌクレオチドタグのループ部分が9ヌクレオチド以上の長さである、請求項128に記載の方法。
- 前記ヘアピンオリゴヌクレオチドタグのループ部分が10ヌクレオチド以上の長さである、請求項128に記載の方法。
- 前記ヘアピンオリゴヌクレオチドタグが一意識別子領域を含む、請求項128に記載の方法。
- 前記一意識別子領域が前記ヘアピンオリゴヌクレオチドタグのループ部分にある、請求項159に記載の方法。
- 前記一意識別子領域が前記ヘアピンオリゴヌクレオチドタグのステム部分にある、請求項159に記載の方法。
- 前記一意識別子領域が前記ヘアピンオリゴヌクレオチドタグのオーバーハング部分にある、請求項159に記載の方法。
- 前記標識が一意識別子領域を含む、請求項133に記載の方法。
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JP2020502255A (ja) * | 2016-09-26 | 2020-01-23 | セルラー リサーチ, インコーポレイテッド | バーコード付きオリゴヌクレオチド配列を有する試薬を用いたタンパク質発現の測定 |
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